Differentiation of hematopoietic stem cells (HSCs) is required for the production and maintenance of all blood cell types for the life of an organism. In people, approximately 1011-1012 new blood cells are produced daily in order to maintain the homeostasis. Aberrant control of hematopoiesis is the underlying cause of many diseases including immune deficiency, anemia, autoimmunity, leukemia, and lymphoma. Basic research over the last 50+ years has allowed for the description of stem cell activity and has facilitated the development of an extremely beneficial treatment - HSC transplantation. A complete understanding of the molecular mechanisms that control hematopoiesis is essential for continued progress in the treatment and/or prevention of hematological malignancies and diseases in people. Progress in the field is also essential to develop more efficient strategies fo HSC transplantation, particularly in adults. We recently showed that the highly conserved 15 kDa Proliferating Cell Nuclear Antigen (PCNA) associated factor (Paf) is a novel and essential HSC cell cycle regulator. In the absence of Paf, HSC function is compromised. In particular, progenitor development is disrupted and there is a reduction in the size of central and peripheral hematopoietic cell compartments. When placed in competition with wild type HSCs, Paf deficient HSC development is obliterated. Therefore, Paf is a key determinant for HSC identity. In this R21 application, we describe experimental systems that will determine the molecular mechanisms by which Paf mediates HSC function and development. While we have shown that Paf can interact with PCNA, it is not known if this interaction actually plays a role in HSC development. PCNA has essential roles in multiple processes, such as DNA replication and repair, cell cycle control, chromatin remodeling, epigenetic inheritance, sister chromatid cohesion, and cell survival. Therefore, it is possible that Paf's primary function is to modify one or more of the functions of PCNA. We will directly test this possibility in Aim 1 by studying mice that express a mutant PAF protein that is unable to interact with PCNA. Our studies have also shown that a significant fraction of PAF purified from cell lysates is modified by the addition of mono- and di-ubiquitin (Ub) on residue K24. The PAF K24 Ub-Ub linkage is mediated via K63 of Ub, which is understood to modify protein function and/or the cellular localization. Aim 2, therefore will focus on the analyses of mice that express a PAF mutant that cannot be K24 Ub modified. These studies will determine if the function of PAF is dependent upon K63-linked Ub mediated interactions with other proteins. A comprehensive analysis of HSC function and development will determine if the two mutant PAF molecules correct all, some or none of the observed HSC defects in Paf-/- mice. The data from these experiments represent an essential step towards delineating the molecular mechanisms by which Paf functions to mediate hematopoiesis.